Enteropathogenic E. coli strains are well known causes of diarrhoea and haemorrhagic colitis in humans and can lead to potentially life threatening sequelae including haemolytic uremic syndrome and thrombotic thrombocytopaenic purpura. Some of these strains are commonly found in livestock and infection in humans is usually a consequence of consumption of contaminated meat or dairy products which have been improperly processed. The O specific polysaccharide component (the “O antigen”) of lipopolysaccharide is known to be a major virulence factor of enteropathogenic E. coli strains.
The E. coli O antigen is highly polymorphic and 166 different forms of the antigen have been defined; Ewing, W. H. [in Edwards and Ewings “Identification of the Enterobacteriacea” Elsevier. Amsterdam (1986)] discusses 128 different O antigens while Lior H. (1994) extends the number to 166 [in “Classification of Escherichia coli In Escherichia coli in domestic animals and humans pp31–72. Edited by C. L. Gyles CAB International]. The genus Salmonella enterica has 46 known O antigen types [Popoff M. Y. et al (1992) “Antigenic formulas of the Salmonella enterica serovars” 6th revision WHO Collaborating Centre for Reference and Research on Salmonella enterica, Institut Pasteur Paris France].
An important step in determining the biosynthesis of O antigens-and therefore the mechanism of the polymorphism has been to characterise the gene clusters controlling O antigen biosynthesis. The genes specific for the synthesis of the O antigen are generally located in a gene cluster at map position 45 minutes on the chromosome of E. coli K-12 [Bachmann, B. J. 1990 “Linkage map of Escherichia coli K-12”. Milcrobiol. Rev. 54:130–197], and at the corresponding position in S. enterica LT2 [Sanderson et al (1995) “Genetic map of Salmonella enterica typhimurium”, Edition VIII Microbiol. Rev. 59: 241–303]. In both cases the O antigen gene cluster is close to the gnd gene as is the case in other strains of E. coli and S. enterica [Reeves P. R. (1994) “Biosynthesis and assemby of lipopolysaccharide, 281–314. in A. Neuberger and L. L. M. van Deenen (eds) “Bacterial cell wall, new comprehensive biochemistry” vol 27 Elsevier Science Publishers]. These genes encode enzymes for the synthesis of nucleotide diphosphate sugars and for assembly of the sugars into oligosaccharide units and in general for polymerisation to O antigen.
The E. coli O antigen gene clusters for a wide range of E. coli O antigens have been cloned but the O7, O9, O16 and O111 O antigens have been studied in more detail with only O9 and O16 having been fully characterised with regard to nucleotide sequence to date [Kido N., Torgov V. I., Sugiyama T., Uchiya K., Sugihara H., Komatsu T., Kato N. & Jann K. (1995) “Expression of the O9 polysaccharide of Escherichia coli: sequencing of the E. coli O9 rfb gene cluster, characterisation of mannosyl transferases, and evidence for an ATP-binding cassette transport system” J. of Bacteriol. 177 2178–2187; Stevenson G., Neal B., Liu D., Hobbs M., Packer N. H., Batley M., Redmond J. W., Lindguist L. & Reeves PR (1994) “Structure of the O antigen of E. coli K12 and the sequence of its rfb gene cluster” J. of Bacteriol. 176 4144–4156; Jayaratne, P. et al. (1991) “Cloning and analysis of duplicated rfbM and rfbK genes involved in the formation of GDP-mannose in Escherichia coli O9:K30 and participation of rfb genes in the synthesis of the group 1 K30 capsular polysaccharide” J. Bacteriol. 176: 3126–3139; Valvano, M. A. and Crosa, J. H.(1989)“Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1”. Inf and Immun. 57:937–943; Marolda C. L. And Valvano, M. A. (1993). “Identification, expression, and DNA sequence of the GDP-mannose biosynthesis genes encoded by the O7 rfb gene cluster of strain VW187 (Eschericia coli O7:K1)”. J. Bacteriol. 175:148–158.1].
Bastin D. A., et al. 1991 [“Molecular cloning and expression in Escherichia coli K-12 of the rfb gene cluster determining the O antigen of an E. coli O111 strain”. Mol. Microbiol. 5:9 2223–2231] and Bastin D. A. and Reeves, P. R. [(1995)“Sequence and analysis of the O antigen gene(rfb)cluster of Escherichia coli O111”. Gene 164: 17–23] isolated chromosomal DNA encoding the E. coli 0111 rfb region and characterised a 6962 bp fragment of E. coli 0111 rfb. Six open reading frames (orfs) were identified in the 6962 bp partial fragment and the alignment of the sequences of these orfs revealed homology with genes of the GDP-mannose pathway, rfbK and rfbM, and other rfb and cps genes.
The nucleotide sequences of the loci which control expression of Salmonella enterica B, A, D1, D2, D3, C1, C2 and E O antigens have been characterised [Brown, P. K., L. K. Romana and P. R. Reeves (1991) “Cloning of the rfb gene cluster of a group C2 Salmonella enterica”: comparison with the rfb regions of groups B and D Mol. Microbiol. 5:1873–1881; Jiang, X.-M., B. Neal, F. Santiago, S. J. Lee, L. K. Romana, and P. R. Reeves (1991) “Structure and sequence of the rfb (O antigen) gene cluster of Salmonella enterica serovar typhimurium (LT2)”. Mol. Microbiol. 5:692–713; Lee, S. J., L. K. Romana, and P. R. Reeves (1992) “Sequences and structural analysis of the rfb (O antigen)gene cluster from a group C1 Salmonella enterica enterica strain” J. Gen. Microbiol. 138: 1843–1855; Lui, D., N. K. Verma, L. K. Romana, and P. R. Reeves (1991) “Relationship among the rfb regions of Salmonella enterica serovars A, B and D” J. Bacteriol. 173: 4814–4819; Verma, N. K., and P. Reeves (1989) “Identification and sequence of rfbS and rfbE, which determine the antigenic specificity of group A and group D Salmonella entericae” J. Bacteriol. 171: 5694–5701; Wang, L., L. K. Romana, and P. R. Reeves (1992) “Molecular analysis of a Salmonella enterica enterica group E1 rfb gene cluster: O antigen and the genetic basis of the major polymorphism” Genetics 130: 429–443; Wyk, P., and P. Reeves (1989). “Identification and sequence of the gene for abequose synthase, which confers antigenic specificity on group B Salmonella entericae: homology with galactose epimerase” J. Bacteriol. 171: 5687–5693,; Xiang, S. H., M. Hobbs, and P. R. Reeves. 1994 Molecular analysis of the rfb gene luster of a group D2 Salmonella enterica strain: evidence for its origin from an insertion sequence-mediated recombination event between group E and D1 strains. J. Bacteriol. 176: 4357–4365; Curd, H., D. Liu and P. R. Reeves, 1998. Relationships among the O antigen Salmonella enterica groups B, D1, D2, and D3. J. Bacteriol. 180: 1002–1007.).
Of the closely related Shigella (which really can be considered to be part of E. coli) S. dysenteriae and S. flexneri O antigens have been fully sequenced and are next to gnd. [Klena JD & Schnaitman CA (1993) “Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1” Mol. Microbiol. 9 393–402; Morona R., Mavris M., Fallarino A. & Manning P. (1994) “Characterisation of the rifc region of Shigella flexneri” J.Bacteriol 176: 733–747].
Inasmuch as the O antigen of enteropathogenic E. coli strains and the O antigen of Salmonella enterica strains are major virulence factors and are highly polymorphic, there is a real need to develop highly specific, sensitive, rapid and inexpensive diagnostic assays to detect E. coli and assays to detect S. enterica. There is also a real need to develop diagnostic assays to identify the O antigens of E. coli strains and assays to identify the O antigens of S. enterica strains. With regard to the detection of E. coli these needs extend beyond EHFC (enteropathogenic haemorrhagic E. coli) strains but this is the area of greatest need. There is interest in diagnostics for ETEC (enterotoxigenic E. coli) etc in E. coli. 
The first diagnostic systems employed in this field used large panels of antisera raised against E. coli O antigen expressing strains or S. enterica O antigen expressing strains. This technology has inherent difficulties associated with the preparation, storage and usage of the reagents, as well as the time required to achieve a meaningful diagnostic result.
Nucleotide sequences derived from the O antigen gene clusters of S. enterica strains have been used to determine S. enterica O antigens in a PCR assay [Luk, J. M. C. et al. (1993) “Selective amplification of abequose and paratose synthase genes (rfb) by polymerase chain reaction for identification of S. enterica major serogoups (A, B, C2, and D)”, J. Clin. Microbiol. 31:2118–2123 ]. The prior complete nucleotide sequence characterisation of the entire rfb locus of serovars Typhimurium, Paratyphi A, Typhi, Muenchen, and Anatum; representing groups B, A, D1, C2 and E1 respectively enabled Luk et al. to select oligonucleotide primers specific for those serogroups. Thus the approach of Luk et al. was based on aligning known nucleotide sequences corresponding to CDP-abequose and CDP-paratose synthesis genes within the O antigen regions of S. enterica serogroups E1, D1, A, B and C2 and exploiting the observed nucleotide sequence differences in order to identify serotype-specific oligonucleotides.
In an attempt to determine the O antigen serotype of a Shiga-like toxin producing E. coli strain, Paton, A. W., et al. 1996 [“Molecular microbiological investigation of an outbreak of Hemolytic-Uremic Syndrome caused by dry fermented sausage contaminated with Shiga-like toxin producing Escherichia coli”. J. Clin. Microbiol. 34: 1622–1627], used oligonucleotides derived from the wbdI (orf6) region, which were believed to be specific to the E. coli O111 antigen and which were derived from E. coli O111 sequence, in a PCR diagnostic assay. Unpublished reports indicate that the approach of Paton et al. is deficient in that the nucleotide sequences derived from wbdI may not specifically identify the O111 antigen and in fact lead to detection of false positive results. Paton et al. disclose the detection of 5 O111 antigen isolates by PCR when in fact from only 3 of those isolates did they detect bacteria which reacted with O111 specific antiserum.